Project Summary The development of chromosome conformation capture techniques such as Hi-C have made it possible to identify domains of interacting chromatin within the nucleus at high resolution across the entire genome. These domains are known as Topologically Associating Domains (TADs) and their boundaries act as insulator elements. While it is now straightforward to identify such domains, there is a gap in knowledge because the genetic control of TAD boundary strength and location is not fully understood, nor is it clear how variation in these boundary properties affects molecular phenotypes such as gene expression. The long-term goal of this applicant?s laboratory is to understand the evolutionary processes that lead to changes in genome organization within and between species. The overall objective of this project is to identify and functionally characterize the genetic determinants of TAD boundary strength within Drosophila melanogaster, as well as the determinants of boundary gain and loss between D. melanogaster and closely related species. Preliminary data produced in the applicant?s laboratory suggests that there is variation in TAD boundary strength among D. melanogaster individuals from the Drosophila Genetic Resource Panel (DGRP) and that novel TAD boundaries are present even between the closely related species of D. melanogaster and D. yakuba. The rationale for the proposed research is that understanding the genetic basis of TAD boundaries will provide insight into how variation in 3D genome organization is related to the emergence of novel phenotypes as well as disease states. The objective of this project will be achieved by pursuing three specific aims: 1) Identify sequence variants that affect TAD boundary strength using association mapping; 2) Determine how TAD boundaries evolve at novel locations in the genome; and 3) Functionally characterize candidate variants using CRISPR in D. melanogaster. The DGRP, which was specifically developed as a resource for mapping quantitative traits, will be used to identify sequence variants associated with TAD boundary strength, whereas 13 Drosophila species from the melanogaster group will be used to identify lineage-specific sequence substitutions involved in the formation of novel TAD boundaries. Candidate causal sequences will be validated using CRISPR genome editing to determine their effect on TAD boundary strength and location. The proposed research is innovative because it represents a substantial departure from the status quo: instead of testing for enrichment of specific genomic features within TAD boundaries, this project proposes to take advantage of natural variation within and between species of Drosophila. One of the major goals of the field is to ultimately predict TAD boundary strength and location from DNA sequence alone. The proposed research is significant because it will bring us closer to this goal by increasing our understanding of how TAD boundaries originate and how they contribute to variation in gene expression levels both within and between species.